The mammalian acrosome reaction (AR) is a sperm head exocytotic event essential to fertilization. A neuronal glycine receptor/Cl channel (GlyR) is present in mammalian sperm and is necessary for the AR initiated by the egg zona pellucida (ZP) protein ZP3. The overall goal of this grant is to increase our understanding of the role of the GlyR in the ZP-initiated AR and the mechanisms involved in controlling its activation. We hypothesize that ZP activates the GlyR directly or via receptor cross-talk, that Cl- efflux through the GlyR is at least partly responsible for depolarization leading to Ca 2+ influx essential to the AR and that activation of the GlyR involves PKA-mediated phosphorylation. The effects of mouse ZP and human recombinant human ZP3 (rhZP3) on mouse sperm and human sperm respectively will be studied using imaging, photometry and antagonists in in vitro AR assays. The following are the Specific Aims. 1. Determination of whether the human and mouse sperm GlyR mediate the intracellular Ca 2+ (Ca2+i) increase elicited by rhZP3, mouse ZP or glycine during the AR. 2. Determination of whether the human and mouse sperm GlyR are involved in the membrane potential change elicited by glycine, rhZP3 or mouse ZP. 3. Determination of whether phosphorylation of the GlyR by protein kinase A (PKA) is involved in AR initiated by glycine, mouse ZP or rhZP3. 4. Determination of whether PKA is important to GlyR mediation of membrane potential change and to the Ca2+i increase elicited by glycine, mouse ZP and rhZP3. 5. Determination of whether mouse ZP-mediated membrane potential and Ca2+i changes would be absent or decreased in sperm from mice with GlyR mutations. 6. Determination of whether the AR of sperm from mice with GlyR mutations occur on the ZP of intact mouse eggs in vitro. If not, determination of whether the AR and fertilization be stimulated by progesterone in these sperm while they are on the ZP? Being able to compare results obtained with human sperm and rhZP3 and those obtained with mouse sperm and mouse ZP synthesized in vivo will help us determine whether common mechanisms are involved in mammalian species.